Peptides are valuable and effective drugs for targeting extracellular receptors. Their use for modulating intracellular processes is hampered by their inability to enter cells, their instability, and their susceptibility to proteases. One effective strategy of stabilizing peptides involves locking them into specific, protease-resistant shapes. The α-helix is one of the major structural components of proteins and is often found at the interface of protein contacts, participating in a wide variety of intermolecular biological recognition events. However, α-helical peptides have a propensity for unraveling and forming random coils, which are, in most cases, biologically less active, or even inactive, and are highly susceptible to proteolytic degradation.
“Peptide stapling” is a term coined for a synthetic methodology used to covalently join two olefin-containing side chains present in a peptide chain using an olefin metathesis reaction (J. Org. Chem. (2001) 66(16); Blackwell et al., Angew. Chem. Int. Ed. (1994) 37:3281). Stapling of a peptide using a hydrocarbon cross-linker created from an olefin metathesis reaction has been shown to help maintain a peptide's native conformation, particularly under physiological conditions (U.S. Pat. No. 7,192,713; Schafineister et al., J. Am. Chem. Soc. (2000) 122:5891-5892; Walensky et al., Science (2004) 305:1466-1470). A staple stabilizes a peptide in a configuration that matches the binding site of the protein target, it protects the peptide against proteolytic action, and it makes the peptide membrane permeable (Sun et al., Biophys. J. 104(9):1923-1932 (2013)). Small molecules are also cell permeable, but they are more limited in the types of targets they can bind. Stapled peptides exhibit higher specificity and affinity than small molecules, targeting intracellular control points that cannot be modulated by current therapeutics.
Protein-protein interactions (PPIs) are involved in many biological processes, hence, the discovery of molecules that perturb PPIs has led to some attractive approaches in drug discovery. PPIs involving an oft-occurring protein α-helix of 1-4 helical turns (4-15 amino acids) are promising targets, because one can design and prepare synthetic α-helix peptide ligands as the receptor antagonist. To enhance the α-helix structural stability of these short synthetic peptides in water, various covalent sidechain-to-sidechain linking strategies have been developed to stabilize the α-helical structure, including KD lactam linkers, hydrocarbon linkers, “click” triazole linkers, m-xylene thioether linkers, perfluorobenzyl thioether linkers, and alkyl thioether linkers (FIGS. 5A-5C and 5E-5G).
Each of these linkers has shown successful applications to some extent. For example, KD lactam-type peptides have been used as HIV and RSV PPI inhibitors, hydrocarbon-type peptides have been developed to promote Bcl2 apoptosis and inhibit HIV-1 capsid assembly or NOTCH transcription, and triazole-type peptides have been applied to PTH and β-catenin/Bcl9.
There are various strategies for generating stapled peptides. The main strategies involve the use of cysteine side chains for forming disulfide bridges and thioether formation (FIGS. 5D and 5H). Other methods involve ring-closing metathesis; biaryl linkage of functionalized synthetic amino acids involving borylated phenylalanine derivatives; or “click chemistry”, whereby cycloaddition between an azide and a terminal or internal alkyne yields a 1,2,3-Triazole (FIG. 5C). These syntheses are expensive and laborious. Owing to the increasing interest in stapled peptides and other conformationally restricted structures, many efforts have been made to develop alternative practical and general preparative methods for their generation. Generally speaking, the above-described linkers are hydrophobic, which often causes solubility problems. Thus, improved compositions and strategies for making stapled peptides are highly desirable.
It is an object of the invention to provide stapled helical peptides with improved properties, such as higher hydrophilicity and/or greater solubility in aqueous solutions.
It is a further object of the invention to provide improved methods of preparing stapled helical peptides.
It is a further object of the invention to provide methods of treatment using stapled helical peptides.